Comparison of Mouse Ly5a and Ly5b Leucocyte Common Antigen Alleles
Comparison of Mouse Ly5a. and Ly5b Leucocyte Common Antigen Alleles
SUZANNE L. ZEBEDEE 0
DIANA S. BARRITT 0
WILLIAM C. RASCHKE 0
0 La Jolla Biological Laboratories, The Salk Institute , P.O. Box 85350, San Diego, California 92186 , USA
The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains. To investigate the divergence between alleles, cDNA clones to the SJA (Ly5a) LCA gene have been isolated and sequenced..A comparison of this information to the Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These base substitutions correspond to five amino-acid changes within the extracellular domain of the LCA molecule. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat LCA sequences. Thus, these two mouse LCA alleles exhibit a pattern of sequence conservation that mimics that found over a much broader scale of evolution. Analysis of antigenicity profiles for each of the allelic sequence changes reveals three molecular domains of altered antigenicity that could account for observed serological differences between the two alleles. Sequence information from the 5' end of the Ly5 LCA gene, generated using polymerase chain-reaction techniques on genomic DNA, reveals eight additional nucleotide differences between the Ly5 and Ly5b alleles.
cDNA; domain; epitopes; evolution
INTRODUCTION
The leucocyte common antigen family of
glycoproteins is abundantly expressed on the surface
of most cells in .the hematopoietic lineage
(Scheid
and Triglia, 1979; Sarmiento et al., 1982)
. These
antigens (also known as CD45, Ly5, T200, and
B220) have been identified in mouse, rat, chicken,
and human systems
(Komuro et al., 1975; Fabre
and Williams, 1977; Judd et al., 1980; Omary et
al., 1980; Houssaint et al., 1987)
. Data from the
analysis of cDNA clones indicate that the murine
LCA molecule has a hydrophobic leader
sequence, an N-terminal extracellular domain
consisting of 402-.541 amino acids, a single
transmembrane region, and a large cytoplasmic
domain of 705 residues
(Saga et al., 1986, with a
correction, 1987; Thomas et al., 1987)
. The
mapping of this family as a single gene on the mouse
chromosome 1 has been completed and shown to
*Corresponding author.
comprise 34 exons
(Saga et al., 1988)
; exons la
and l b are alternatively excluded 5? untranslated
sequences of LCA mRNAs, and exons 2-33 are
protein-encoding. The function of the leucocyte
common antigen has not been determined,
although its involvement in leucocyte activities,
such as natural killer cytolysis (Sparrow and
McKenzie, 1983), cytotoxic T-cell cytolysis
(Harp
et al., 1984)
, as well as lymphocyte activation and
differentiation
(Yakura et al., 1986; Mittler et al.,
1987; Pingel and Thomas, 1989)
has been
implicated. These roles may be mediated through the
tyrosine phosphatase activity shown to be
associated with the LCA molecule
(Tonks et al., 1988)
.
In the mouse, the LCA molecule shows
heterogeneity in molecular weight, glycosylation, and
antigenicity patterns. Differences in LCA family
members have been traced to the alternative
splicing of exons 4, 5, and 6, which generates
multiple molecular isoforms
(Saga et al., 1987;
Thomas et al., 1987)
. In B lymphoid cells, the
major LCA protein product is 220,000 m.w. and
contains all three of these alternative exons,
whereas the major thymocyte protein is 180,000
m.w. and does not include exons 4, 5, or 6. These
differences account for an insertion of 139
residues in the extracellular domain of the B-cell
expressed LCA molecule. Proteins of
intermediate size have been observed, which apparently
represent splice combinations of exons 4, 5, and 6
(Chang et al., 1989)
. Glycosylation differences in
the LCA protein family are also due to the
inclusion of these variable exon sequences as the
inserted amino acids contain many serine and
threonine residues potentially linked to O-type
carbohydrate structures
(Childs et al., 1983;
Johnson et al., 1989)
.
Murine LCA glycoproteins bear the Ly5
alloantigenic determinant, which has been
characterized in both inbred and wild strains of mice
(Seldin et al., 1987)
. Inbred mice have been
categorized into three LCA alleles, Ly5a, Ly5b, and
Ly5c, which can be distinguished antigenically by
their reactivity with specific monoclonal and
alloantibodies (Ly5.1, Ly5.2) and genetically by
RFLPs of mouse genomic DNA
(Seldin et al.,
1987)
. Most established murine strains express
the Ly5b allele, including BALB/c, C57BL,
CBA/J, and NZB/BLNJ, yet SJL/J mice carry the
Ly5 allele.
To examine the differences between the murine
Ly5 and Ly5b alleles, we have isolated and
analyzed cDNA clones from t (...truncated)